Cytocompatible alginate gels

ABSTRACT

The present invention relates to a method of making cytocompatible alginate gels and their use in the treatment of cardiomyopathy.

FIELD OF THE INVENTION

This invention relates to the fields of organic chemistry, polymerscience, material science and medical science. In particular, it relatesto a method of making a cytocompatible alginate gel composition forcellular treatment of cardiovascular diseases, in particularcardiomyopathy.

BACKGROUND OF THE INVENTION

Ischemic heart disease typically results from an imbalance between themyocardial blood flow and the metabolic demand of the myocardium.Progressive atherosclerosis with increasing occlusion of coronaryarteries leads to a reduction in coronary blood flow. Atherosclerosis isa type of arteriosclerosis in which cells including smooth muscle cellsand macrophages, fatty substances, cholesterol, cellular waste product,calcium and fibrin build up in the inner lining of a body vessel.Arteriosclerosis is thickening and hardening of arteries. Blood flow canbe further decreased by additional events such as changes in circulationthat lead to hypoperfusion, vasospasm or thrombosis.

Myocardial infarction (MI) is one form of heart disease that oftenresults from the sudden lack of supply of oxygen and other nutrients.The lack of blood supply is a result of a closure of the coronary artery(or any other artery feeding the heart) which nourishes a particularpart of the heart muscle. The cause of this event is generallyattributed to arteriosclerosis in coronary vessels.

Formerly, it was believed that an MI was caused by a slow progression ofclosure from, for example, 95% then to 100%. However, an MI can also bea result of initially minor blockages where, for example, there is arupture of a cholesterol plaque, subsequent formation of blood clots inthe artery resulting in blockage of the flow of blood and eventualdownstream cellular damage. This damage can cause irregular rhythms thatcan be fatal, even though the remaining muscle is strong enough to pumpa sufficient amount of blood. As a result of this insult to the hearttissue, scar tissue tends to naturally form.

Various procedures, including mechanical and medicinal, are known forreopening blocked arteries. An example of a mechanical procedure isballoon angioplasty with stenting, while an example of a medicinaltreatment is the administration of a thrombolytic agent, such asurokinase. Such procedures do not, however, treat actual tissue damageto the heart. Other systemic drugs, such as ACE-inhibitors andbeta-blockers, may be effective in reducing cardiac load post-MI,although a significant portion of the population that experiences amajor MI ultimately develop heart failure.

An important component in the progression to heart failure is post-MIremodeling of the heart due to mismatched mechanical forces between theinfarct region and the healthy tissue resulting in uneven stressdistribution in the wall of the left ventricle. The principle componentsof remodeling include myocyte death, edema and inflammation, followed byfibroblast infiltration and collagen deposition, and finally scarformation. The main component of the scar is collagen. Since maturemyocytes of an adult are not regenerated, the infarct region experiencessignificant thinning. Myocyte loss is the major etiologic factor of wallthinning and chamber dilation that may ultimately lead tocardiomyopathy. Further, remote regions of the heart may experiencehypertrophy (thickening) resulting in an overall enlargement of the leftventricle. These changes in the heart often result in changes in apatient's lifestyle, e.g., the ability to walk and to exercise. Thesechanges also correlate with physiological changes that result inincrease in blood pressure and worsening systolic and diastolicperformance.

Another means of treating post-MI complications such as cardiomyopathyis dynamic cardiomyoplasty wherein a patient's latissimus dorsi muscleis wrapped around the ventricles and electostimulated in synchrony withthe contractions of the heart by means of an implantedcardio-myostimulator. A relatively recent modification of dynamiccardiomyoplasty is cellular cardiomyoplasty in which individual cellsare delivered to the damaged myocardium where they integrate into themyocardial tissue proliferate and eventually provide an improvement incontractile force. The cells may be, without limitation, fetal orembryonic cardiomyocytes, adult cardiomyocytes, skeletal myoblasts,smooth muscle cells, bone marrow derived stromal cells, undifferentiatedblood cells and the like. The problem is delivering the cells to thedamaged myocardium and retaining them there in a fully operativecondition until the cells have had the opportunity to achieve theirtherapeutic effect either by secretion of a plethora of cytokines, or byintegrating into the heart tissue and or/by trans-differentiating intocardiomyocytes.

What is needed, then, is a method of delivering cells to the damagedmyocardium and retaining the cells at the myocardial locus in theirnative fully potent state until they have had the opportunity to exerttheir therapeutic benefit. The current invention provides such a methodand a composition to use with the method.

SUMMARY OF THE INVENTION

Thus, in one aspect, the current invention relates to a method offorming a cytocompatible alginate gel, comprising:

-   providing an alginate;-   dispersing substantially homogeneously within the alginate a    plurality of cells;-   dispersing substantially homogeneously within the alginate a    plurality of vesicles; and-   adding to the alginate a divalent metal cation to form an alginate    gel.

In an aspect of this invention, the plurality of cells are selected fromthe group consisting of localized cardiac progenitor cells, mesenchymalstem cells, bone marrow derived mononuclear cells, adipose tissuederived stem cells, embryonic stem cells, umbilical cord blood derivedstem cells, smooth muscle cells and skeletal myoblasts.

In an aspect of this invention, the plurality of vesicles are selectedfrom the group consisting of liposomes and polymerosomes and othermultilamellar vesicles.

In an aspect of this invention, the divalent metal cation comprisesCa⁺⁺.

In an aspect of this invention, the vesicles comprise one or moresubstances that are released from the vesicles and that thereuponenhance the cytocompatibility of the alginate gel that is formed.

In an aspect of this invention, the one or more substances comprisegrowth factors.

In an aspect of this invention, the growth factors are selected from thegroup consisting of isoforms of vasoendothelial growth factor (VEGF),fibroblast growth factor (FGF, e.g. beta-FGF), Del 1, hypoxia inducingfactor (HIF 1-alpha), monocyte chemoattractant protein (MCP-1),nicotine, platelet derived growth factor (PDGF), insulin-like growthfactor 1 (IGF-1), transforming growth factor (TGF alpha), hepatocytegrowth factor (HGF), estrogens, Follistatin, Proliferin, ProstaglandinE1 and E2, tumor necrosis factor (TNF-alpha), Interleukin 8 (II-8),hematopoietic growth factors, erythropoietin, granulocyte-colonystimulating factors (G-CSF) and platelet-derived endothelial growthfactor (PD-ECGF).

In an aspect of this invention, the one or more substances comprise celladhesion proteins.

In an aspect of this invention, the cell adhesion proteins are selectedfrom the group consisting of laminin, fibronectin, RGD, synthetic RGDand cyclic RGD (c-RGD).

In an aspect of this invention, a composition comprising an alginate gelprepared by the above method.

An aspect of this invention is a method of forming a cytocompatiblealginate gel, comprising:

-   providing an alginate;-   dispersing substantially homogeneously within the alginate a    plurality of cells;-   dispersing substantially homogeneously within the alginate a    micronized tissue construct; and-   adding to the alginate a divalent metal cation to form an alginate    gel.

In an aspect of this invention, the plurality of cells are selected fromthe group consisting of localized cardiac progenitor cells, mesenchymalstem cells, bone marrow derived mononuclear cells, adipose tissuederived stem cells, embryonic stem cells, umbilical cord blood derivedstem cells, smooth muscle cells and skeletal myoblasts.

In an aspect of this invention, the micronized tissue construct areselected from the group consisting of urinary bladder matrix (UBM) andsmall intestinal sub-mucosa (SIS).

In an aspect of this invention, the divalent metal cation comprisesCa⁺⁺.

In an aspect of this invention, a composition comprising acytocompatible alginate gel prepared by the above method.

An aspect of this invention is a method of forming a cytocompatiblealginate gel, comprising:

-   providing an alginate;-   dispersing substantially homogeneously within the alginate a    plurality of cells;-   dispersing substantially homogeneously within the alginate a    plurality of nano or microparticles, wherein:

the microparticles comprise an alginate lyase; and

-   a divalent metal cation to form an alginate gel.

In an aspect of this invention, the plurality of cells are selected fromthe group consisting of localized cardiac progenitor cells, mesenchymalstem cells, bone marrow derived mononuclear cells, adipose tissuederived stem cells, embryonic stem cells, umbilical cord blood derivedstem cells, smooth muscle cells and skeletal myoblasts.

In an aspect of this invention, the divalent metal cation comprisesCa⁺⁺.

In an aspect of this invention, the nano or microparticles comprisealginate lyases.

In an aspect of this invention, a composition comprising an alginate gelprepared by the above method.

An aspect of this invention is a method of forming a cytocompatiblealginate gel, comprising:

-   providing an alginate;-   dispersing substantially homogeneously within the alginate a    plurality of cells;-   dispersing substantially homogeneously within the alginate an    alginate lyase; and-   adding to the alginate a divalent metal cation to form an alginate    gel.

In an aspect of this invention, the plurality of cells are selected fromthe group consisting of localized cardiac progenitor cells, mesenchymalstem cells, bone marrow derived mononuclear cells, adipose tissuederived stem cells, embryonic stem cells, umbilical cord blood derivedstem cells, smooth muscle cells and skeletal myoblasts.

In an aspect of this invention, the divalent metal cation comprisesCa⁺⁺.

In an aspect of this invention, a composition comprising an alginate gelprepared by the above method.

An aspect of this invention is a method of forming acytocompatiblealginate gel, comprising:

-   providing an alginate;-   dispersing substantially homogeneously within the alginate a    plurality of cells;-   dispersing substantially homogeneously within the alginate a    PEGylated alginate lyase within a degradable microsphere; and-   adding to the alginate a divalent metal cation to form an alginate    gel.

In an aspect of this invention, the plurality of cells are selected fromthe group consisting of localized cardiac progenitor cells, mesenchymalstem cells, bone marrow derived mononuclear cells, adipose tissuederived stem cells, embryonic stem cells, umbilical cord blood derivedstem cells, smooth muscle cells and skeletal myoblasts.

In an aspect of this invention, the divalent metal cation comprisesCa⁺⁺.

In an aspect of this invention, a composition comprising an alginate gelprepared by the above method.

An aspect of this invention is a method of forming a cytocompatiblealginate gel, comprising:

-   providing an alginate;-   subjecting to a treatment that lowers the molecular weight of the    alginate;-   dispersing substantially homogeneously within the alginate a    plurality of cells; and-   adding to the alginate a divalent metal cation to form an alginate    gel.

In an aspect of this invention, the plurality of cells are selected fromthe group consisting of localized cardiac progenitor cells, mesenchymalstem cells, bone marrow derived mononuclear cells, adipose tissuederived stem cells, embryonic stem cells, umbilical cord blood derivedstem cells, smooth muscle cells and skeletal myoblasts.

In an aspect of this invention, the divalent metal cation comprisesCa⁺⁺.

In an aspect of this invention, a composition comprising an alginate gelprepared by the above method.

An aspect of this invention is a method of forming a cytocompatiblealginate gel, comprising:

-   providing an alginate;-   subjecting the alginate to oxidation;-   dispersing substantially homogeneously within the alginate a    plurality of cells; and-   adding to the alginate a divalent metal cation to form an alginate    gel.

In an aspect of this invention, the plurality of cells are selected fromthe group consisting of localized cardiac progenitor cells, mesenchymalstem cells, bone marrow derived mononuclear cells, adipose tissuederived stem cells, embryonic stem cells, umbilical cord blood derivedstem cells, smooth muscle cells and skeletal myoblasts.

In an aspect of this invention, the divalent metal cation comprisesCa⁺⁺.

In an aspect of this invention, a composition comprising an alginate gelprepared by the above method.

An aspect of this invention is a method of treating a disease in apatient in need thereof, comprising deploying a cytocompatible alginategel at or near a site where the disease is occurring or is suspected mayoccur.

In an aspect of this invention, the disease is myocardial infarction(MI).

In an aspect of this invention, the disease is myocardial ischemia.

In an aspect of this invention, the disease is myocarditis.

In an aspect of this invention, the disease is cardiomyopathy.

DETAILED DESCRIPTION OF THE INVENTION

Use of the singular herein includes the plural and visa versa unlessexpressly stated to be otherwise. That is, “a” and “the” refer to one ormore of whatever the word modifies. For example, “a liposome” or “apolymerosome” includes one such particle, two such particles or a largeplurality of such particles. Likewise, “a divalent metal cation” or “thedivalent cation” may refer to one, two or plethora of such cations, andso on.

As used herein, “substantial” or “substantially” refers to a conditionthat when so modified is understood to not necessarily be absolute orperfect but would be considered close enough to those of ordinary skillin the art to warrant designating the condition as being present. As anon-limiting example, if cells or vesicles are characterized as being“substantially homogenously” dispersed in a medium, 80% or morehomogeneous dispersion would be understood by one of ordinary skill inthe art to fulfill the requirement.

As used herein, “homogeneous” or “homogeneously” refers to a solution orlayer in which a solute or dispersant is uniformly dispersed throughouta dispersing medium such that a sample taken from anywhere in thesolution or the layer will have the same composition as a sample takenfrom anywhere else in the solution or layer.

As used herein, “dispersing” refers to distribution of one material suchas, without limitation, cells, vesicles or particles in a chosen medium.

As used herein, a “gel” or “hydrogel” refers to a water-insolublesubstance that nevertheless is capable of imbibing a substantial amountof water, the substance swelling in the process. Thus, the “alginategel” of the present invention comprises alginate that has been tonicallycross-linked to render the alginate water insoluble. The alginatenevertheless retains an affinity for water and absorbs substantialquantities of it while not actually dissolving in the water.

As used herein, “alginate” refers to a linear polysaccharide derivedfrom seaweed. The most common source of alginate is the speciesMacrocystis pyrifera. Alginate is composed of repeating units ofD-mannuronic (M) and L-guluronic acid (G), presented in both alternatingblocks and alternating individual residues. Soluble alginate may be inthe form of mono-valent salts including, without limitation, sodiumalginate, potassium alginate and ammonium alginate.

As used herein, a “vesicle” refers to a microscopic particle having ahollow core enclosed within a shell-like structure. The shell may beunilamellar or multilamellar, that is, it may consist of one layer ormultiple layers such as the layers of an onion. The hollow core may befilled only with air or it may be filled with a liquid. Examples ofvesicles useful in this invention include liposomes, polymerosomes andany other unilamellar or multilamellar particle that exhibits therequisite characteristics described below.

As used herein, a “liposome” refers to a vesicle consisting of anaqueous core enclosed by one or more phospholipid layers. Liposomes maybe unilamellar, composed of a single bilayer, or they may bemultilamellar, composed of two or more concentric bilayers. Liposomesrange from about 20-100 nm diameter for small unilamellar vesicles(SUVs), about 100-5000 nm for large multilamellar vesicles andultimately to about 100 microns for giant multilamellar vesicles (GMVs).LMVs form spontaneously upon hydration with agitation of dry lipidfilms/cakes which are generally formed by dissolving a lipid in anorganic solvent, coating a vessel wall with the solution and evaporatingthe solvent. Energy is then applied to convert the LMVs to SUVs, LUVs,etc. The energy can be in the form of, without limitation, sonication,high pressure, elevated temperatures and extrusion to provide smallersingle and multi-lamellar vesicles. During this process some of theaqueous medium is entrapped in the vesicle. Generally, however, thefraction of total solute and therefore the amount of therapeutic agententrapped tends to be rather low, typically in the range of a fewpercent. Recently, liposome preparation by emulsion templating (Pautot,et al., Langmuir, 2003, 19:2870) has been described. Emulsion templatingcomprises, in brief, the preparation of a water-in-oil emulsionstabilized by a lipid, layering of the emulsion onto an aqueous phase,centrifugation of the water/oil droplets into the water phase andremoval of the oil phase to give a dispersion of unilamellar liposomes.This method can be used to make asymmetric liposomes in which the innerand outer monolayers of the single bilayer contain different lipids.Liposomes prepared by any method, not merely those described above, maybe used in the compositions and methods of this invention. Any of thepreceding techniques as well as any others known in the art or as maybecome known in the future may be used as compositions of therapeuticagents in or on a delivery interface of this invention. Liposomescomprising phospho-and/or sphingolipids may be used to deliverhydrophilic (water-soluble) or precipitated therapeutic compoundsencapsulated within the inner liposomal volume and/or to deliverhydrophobic therapeutic agents dispersed within the hydrophobic bilayermembrane.

Polymerosomes can be prepared in the same manner as liposomes. That is,a film of a diblock copolymer can be formed by dissolving the copolymerin an organic solvent, applying a film of the copolymer-containingsolvent to a vessel surface, removing the solvent to leave a film of thecopolymer and then hydrating the film. This procedure, however, tends toresult is a polydispersion of micelles, worm micelles and vesicles ofvarying sizes. Polymerosomes can also be prepared by dissolving thediblock copolymer in a solvent and then adding a poor solvent for one ofthe blocks, which will result in the spontaneous formation ofpolymerosomes.

As with liposomes, polymerosomes can be used to encapsulate therapeuticagents by including the therapeutic agent in the water used to rehydratethe copolymer film. Polymerosomes can also be force-loaded byosmotically driving the therapeutic agent into the core of the vesicle.Also as with liposomes, the loading efficiency is generally low.Recently, however, a technique has been reported that providespolymerosomes of relative monodispersivity and high loading efficiency;generation of polymerisomes from double emulsions. Lorenceau, et al.,Langmuir, 2005, 21:9183-86. The technique involves the use ofmicrofluidic technology to generate double emulsions consisting of waterdroplets surrounded by a layer of organic solvent. Thesedroplet-in-a-drop structures are then dispersed in a continuous waterphase. The diblock copolymer is dissolved in the organic solvent andself-assembles into proto-polymerosomes on the concentric interfaces ofthe double emulsion. The actual polymerosomes are formed by completelyevaporating the organic solvent from the shell. Using this procedure thesize of the polymerosomes can be finely controlled and, in addition, theability to maintain complete separation of the internal fluids from theexternal fluid throughout the process allows extremely efficientencapsulation. This technique along with any other technique known inthe art or as may become known in the future can be used to prepare acomposition of therapeutic agents for use in or on a delivery interfaceof this invention.

As used herein, a “divalent metal cation” refers to a positively chargedion of any metallic element of the Periodic Table having a valence oftwo. Representative examples of divalent metal cations include, but arenot limited to, Ca⁺², Sr⁺², Ba⁺², Mg⁺², Fe⁺², Mn⁺², Cu⁺², Pb⁺², Ni⁺²,Co⁺² and Zn⁺².

As used herein, “cytocompatible” or “cytocompatibility” refers to theability of a carrier substance, herein an alginate gel, to co-exist fora substantial period of time with a variety of cells dispersed withinthe substance without the substance having any deleterious effect on thecells, that is, without limitation, any negative effect on theirrobustness, viability, morphology, physiology, their ability to grow,their ability to proliferate, their ability to express whatevercytokines they normally express in their natural environment and thelike. For the purposes of the current invention a substantial period oftime constitutes at least 14 days.

Heart diseases include, but are not limited to, myocardial infarction,myocardial ischemia, myocarditis, or cardiomyopathy.

As used herein, “myocardial infarction (MI)” refers to the death of ordamage to part of the heart muscle due to an insufficient blood supply.

As used herein, “myocardial ischemia” refers to deficient blood flow topart of the heart muscle.

As used herein, “myocarditis” refers to the inflammation of the heartmuscle (myocardium)

As used herein, “cardiomyopathy” refers to a disease of heart muscles inwhich the heart muscles become inflamed.

Endogenous cardiomyocyte (myocytes) apoptosis is the major etiologicalfactor of wall thinning and chamber dilation and may ultimately lead toprogression of cardiomyopathy. After an infarction, mature myocytes ofan adult are not regenerated which can lead to significant thinning inthe infarct region. Thus, factors which promote cell survival of cellsapplied to the infarct region should be beneficial. In some embodiments,cell survival promoting factors include growth factors such asinsulin-like growth factor (IGF-1) and human growth factor (HGF), whichare known to mediate cell growth, differentiation and survival of avariety of cell types. In addition, small molecules such as HMG-CoAreductase inhibitors (statins) and capsase inhibitors can also promotecell survival and inhibit apoptosis.

To assist in the generation of new cells at the infarct region,autologous or allogeneic stem cells may be delivered to a patient. Asused herein, “autologous” refers to the donor and recipient of the stemcells being the same, i.e., the patient him/herself is the source of thecells. As used herein, “allogeneic” refers to the donor and recipient ofthe stem cells being different individuals. Cell survival promotingfactors can be used to increase the survivability of autologous andallogeneic implanted stem cells at the infarct region.

Cardiac progenitor cells are highly specialized stem cells which haveshown the ability to differentiate into certain types of fully maturecardiac tissue. Examples of cardiac progenitor cells include, but arenot limited to, c-Kit(+), Sca-1(+) and lsl-1(+). Factors which recruitendogenous factors when applied to the infarct region should also bebeneficial. In some embodiments, the endogenous recruiting factor caninclude hepatocyte growth factor (HGF). HGF has been shown to controlcell motility and promote cell migration. If applied post-infarction,HGF can assist in mobilizing and recruiting resident cardiac progenitorcells to the infarct region. In some embodiments, the endogenousrecruiting factor can include stromal cell-derived factor 1 (SDF-1).SDF-1 is the ligand for the CXCR4 receptor, which is a surface receptoron circulating endothelial progenitor cells. When applied in or aroundthe infarct region, SDF-1 may facilitate the homing of circulatingendothelial progenitor cells to induce neovascularization.

While alginate gels increase cell retention at the target site whenco-injected with cells in vitro and in vivo, the cells are viable foronly around 14 days. The cytocompatibility of alginate gels is limitedbecause of very small pore size (40-200 nm) which does not allowspreading of the cells which are usually in the range of 5-40 microns insize depending upon the cell type. However, the pore size is still largeenough to allow nutrient transfer to the cells, hence the observedperiod of viability.

A relatively high concentration of calcium ions (at least 40 mM) isrequired to effect the gelation of alginate (at 0.5% w/v solution).Excess calcium, however, is detrimental to cells and can potentiallycause arrhythmias when injected into the myocardium. The alginate gelsare cross-linked with the calcium ions, which cross-links cannot bedegraded by cellular enzymes to allow cell migration. A larger poresize, one that would permit migration and proliferation of the cells,should prove extremely beneficial.

In an embodiment of this invention, cells and vesicles are homogeneouslydispersed in an alginate followed by addition of a divalent metalcation. A presently preferred divalent metal cation is Ca⁺⁺. Thevesicles of the present invention include, but are not limited to,liposomes, polymerosomes or other multilamellar vesicles. The vesiclesare transformed from a spherical structure to a cochlear structure inthe presence of Ca⁺⁺. When the calcium chloride is added to the alginatedispersion, the calcium ions first crosslink the alginate. Excesscalcium ions are then quenched by the liposomes, polymerosomes or othermultilamellar vesicles to form cochlear structures. The result is moreopen pore space in the alginate gel as the large spatial volumesoccupied by the vesicles prior to contact with Ca⁺⁺ remain after thevesicles collapse into condensed cochlear structures. Thus, the largerpores in the alginate gels are conducive to cell migration and cellproliferation. In addition, the quenching of the calcium ions by thevesicles is beneficial to prevent arrhythmias.

In another embodiment, the vesicles comprise one or more substances thatare released from the vesicles. These substances include, but notlimited to, growth factors. The growth factors are selected from thegroup consisting of isoforms of vasoendothelial growth factor (VEGF),fibroblast growth factor (FGF, e.g. beta-FGF), Del 1, hypoxia inducingfactor (HIF 1-alpha), monocyte chemoattractant protein (MCP-1),nicotine, platelet derived growth factor (PDGF), insulin-like growthfactor 1 (IGF-1), transforming growth factor (TGF alpha), hepatocytegrowth factor (HGF), estrogens, Follistatin, Proliferin, ProstaglandinE1 and E2, tumor necrosis factor (TNF-alpha), Interleukin 8 (II-8),hematopoietic growth factors, erythropoietin, granulocyte-colonystimulating factors (G-CSF) and platelet-derived endothelial growthfactor (PD-ECGF). The growth factors which are released from thevesicles further enhance the cytocompatibility of the alginate gel thatis formed.

In a further embodiment, the vesicles comprise cell adhesion proteins.The cell adhesion proteins are selected from the group consisting oflaminin, fibronectin, RGD, synthetic RGD and cyclic RGD (c-RGD). Thepolypeptide Arg-Gly-Asp (RGD) has been demonstrated to be a bioactivefactor for human endothelial cell attachment and therefore would beexpected to assist in the attachment of cells of this invention as well.In addition to RGD itself, cyclic RGD (cRGD) and RGD mimetics and smallmolecules capable of binding as does RGD to other adhesion receptorsdifferentially expressed on the endothelial cells are within the scopeof this invention. Some examples of cRGD or RGD mimetics include V3antagonists such as IIb/IIIb antagonists (B. S. Coller, Thromb. Haemost.2001, 86:427-443 (Review)), such as abciximax (R. Blindt, J. Mol. Cell.Cardiol. 2000, 32:2195-2206), XJ 735 (S. S. Srivastva et al.,Cardiovasc. Res. 1997, 36:408-428), anti-₃-integrin antibody F11, cRGD(M. Sajid et al., Am. J. Physiol. Cell Physiol., 2003, 285:C1330-1338),and other sequences such as laminin derived SIKVAV (M. H. Fittkau etal., Biomaterials, 2005, 26:167-174), laminin derived YIGSR (S.Kouvroukoglou et al., Biomaterials, 2000, 21:1725-1733), KQAGDV, andVAPG (B. K. Mann, J. Biomed. Mater. Res. 2002, 60:86-93).

In yet another embodiment, an alginate is homogeneously dispersed withcells and micronized tissue construct followed by addition of thedivalent metal cation. The micronized tissue constructs are selectedfrom the group consisting of urinary bladder matrix (UBM) and smallintestinal sub-mucosa (SIS). The UBM or SIS is ground and sieved toachieve a sub 100 micron particle size. This micronized tissue is addedto an alginate solution (Pronova LVG or LVM, 0.2 to 1.0% solids in PBS,preferably 0.5%), at a level of 0.1 to 3% by weight of total solution.The micronized tissue are dispersed throughout the alginate and thecells. The cells secrete proteases to degrade the urinary bladder matrix(UBM) and small intestinal sub-mucosa (SIS). The voids left by thedegraded UBM or SIS can provide spreading room for the cells.

In an embodiment, an alginate is homogeneously dispersed with cells andnano or microparticles comprising an enzyme and the divalent metalcation. The one or more enzymes are selected from the group consistingof alginate lyases. The alginate lyases cause the breakdown of thealginate as it is being cross-linked resulting in larger open spaces inthe gel when it forms. These larger spaces are compatible with cellgrowth and proliferation and thus the procedure should enhance thecytocompatibility of the alginate gel with the cells.

As used herein, “PEG” refers to polyethylene glycol (PEG).

As used herein, “PEGylation” or “PEGylated” refers to a process ofattaching the PEG to other molecules. To couple PEG to a molecule, thePEG is first activated by preparing a derivative of the PEG that resultsin the PEG having a good leaving group at one end. The molecule to bePEGylated must have an active functional group capable of displacing theleaving group.

In one embodiment, an alginate is homogeneously dispersed with cells,followed by addition of the divalent metal cation and a suspension ofdegradable microspheres (e.g. PLGA) containing PEGylated alginate lyase.The PEGylation of an alginate lyase lengthens its circulation time andinhibits its degradation by proteases.

In another embodiment, an alginate is subjected to a treatment thatlowers the molecular weight of the alginate, and then homogeneouslydispersed with a plurality of cells followed by the addition of thedivalent metal cation, when a cytocompatible alginate gel is formed. Thevarious treatments which reduce molecular weight of an alginate include,but are not limited to, gamma irradiation, e-beaming, UV irradiation orcombinations thereof. The alginate may be treated dry or in solution.The reduction in the molecular weight of the alginate by these meanswould have the same beneficial effect in terms of creating larger voidsin the alginate gel as the above discussed lyase addition.

In further embodiment, an alginate is subjected to an oxidation thatpartially oxidizes the alginate, and then homogeneously dispersed withcells, followed by addition of the divalent metal cation, when acytocompatible alginate gel is formed. The oxidation would result in amore hydrolytically labile polymer chain and should result again in theformation of larger voids or pores in the alginate gel with resultantincrease in cytocompatibility of the alginate gel that is formed.

While particular embodiments of the present invention have been shownand described, it will be obvious to those skilled in the art thatchanges and modifications can be made without departing from thisinvention in its broader aspects. Therefore, the appended claims are toencompass within their scope all such changes and modifications as fallwithin the true spirit and scope of this invention.

1. A method of forming a cytocompatible alginate gel, comprising:providing an alginate solution; dispersing within the alginate solutiona plurality of cells and a plurality of vesicles to form a substantiallyhomogenous alginate-cell-vesicle mixture; and adding at least 40 mM of adivalent metal cation to the substantially homogeneousalginate-cell-vesicle mixture to form a cytocompatible alginate gel suchthat the excess amount of the divalent metal cation causes the pluralityof vesicles to form pores within the cytocompatible alginate gel thatare conducive to cell migration and proliferation.
 2. The method ofclaim 1, wherein the plurality of cells are selected from the groupconsisting of: localized cardiac progenitor cells, mesenchymal stemcells, bone marrow derived mononuclear cells, adipose tissue derivedstem cells, embryonic stem cells, umbilical cord blood derived stemcells, smooth muscle cells and skeletal myoblasts.
 3. The method ofclaim 1, wherein the plurality of vesicles are selected from the groupconsisting of: liposomes, polymerosomes and other multilamellarvesicles.
 4. The method of claim 1, wherein the divalent metal cation isCa²⁺.
 5. The method of claim 1, wherein the vesicles comprise one ormore substances that are released from the vesicles and that thereuponenhance the cytocompatibility of the alginate gel that is formed.
 6. Themethod of claim 5, wherein the one or more substances comprise growthfactors.
 7. The method of claim 6, wherein the growth factors areselected from the group consisting of: isoforms of vasoendothelialgrowth factor (VEGF), fibroblast growth factor (FGF, e.g. beta-FGF), Del1, hypoxia inducing factor (HIF 1-alpha), monocyte chemoattractantprotein (MCP-1), nicotine, platelet derived growth factor (PDGF),insulin-like growth factor 1 (IGF-1), transforming growth factor (TGFalpha), hepatocyte growth factor (HGF), estrogens, Follistatin,Proliferin, Prostaglandin E 1 and E2, tumor necrosis factor (TNF-alpha),Interleukin 8 (I1-8), hematopoietic growth factors, erythropoietin,granulocyte-colony stimulating factors (G-CSF) and platelet-derivedendothelial growth factor (PD-ECGF).
 8. The method of claim 5, whereinthe one or more substances comprise cell adhesion proteins.
 9. Themethod of claim 8, wherein the cell adhesion proteins are selected fromthe group consisting of: laminin, fibronectin, RGD, synthetic RGD andcyclic RGD (c-RGD).
 10. A composition comprising an alginate gelprepared by the method of claim
 1. 11. The method of claim 1, whereinthe alginate solution has been subjected to a treatment to lower itsmolecular weight prior to adding the cells and vesicles, wherein thetreatment comprises UV irradiation, gamma irradiation or e-beaming.